Adipocyte differentiation-associated gene and protein

ABSTRACT

Polynucleotides which are produced by preadipocytes within 12 hours from the start of adipocyte differentiation induction and comprise a base sequence identical or at least 93% homologous to SEQ ID NO:1, or a base sequence identical to SEQ ID NO:9, or polynucleotides complementary to those polynucleotides, and proteins comprising an amino acid sequence identical or at least 96% homologous to the protein shown under SEQ ID NO:2 or comprising the amino acid sequence shown under SEQ ID NO:10 are provided.

TECHNICAL FILED

This invention relates to a polynucleotide designated as “clone 24polynucloetide”, which is expressed in the early stage of adipocytedifferentiation and, a protein designated as “clone 24 protein” and,further, to an antibody, an antagonist and an agonist to or of the clone24 protein, to the production of these, and to a pharmaceuticalcomposition and a diagnostic agent containing any of the substancesenumerated above.

BACKGROUND ART

Obesity resulting from supernutrition is now the most important factorcausing such severe lifestyle-related diseases as diabetes, hypertensionand arteriosclerosis. In considering the future life sciences and healthsciences, the elucidation of the molecular mechanisms of obesity may besaid to be an essential task. It is adipocytes that are directlyinvolved in obesity, and the elucidation of the process of adipocytegeneration, if given, will have a direct bearing on the treatment ofobesity. With the recent advances in molecular biology, the mechanismsof adipocyte differentiation are being elucidated. It is known thatthere are a group of key genes and that these form an ingeniousinformation-exchanging network.

The adipocyte differentiation process comprises a complicated series ofsteps, and adipoblasts differentiate into preadipocytes, which thendifferentiate into adipocytes. Transcription factors closely related toadipocyte differentiation have recently been discovered in systems usingcultured cells or genetically modified individuals, for instance.

PPARs (peroxisome proliferator-activated receptors), the C/EBP(CCAAT/enhancer-binding protein) family and SREBP-1/ADD1 (sterolregulatory element binding protein 1 or adipocyte determination anddifferentiation-dependent factor 1) are said to be most importanttranscription factors in adipocyte differentiation.

It has been made clear that PPARs form a family, in which PPARγ isespecially important for adipocyte differentiation. Thus, it has beenmade clear that ectopic expression of PPARγ results in differentiationof adipoblasts and preadipocytes into adipocytes. This result may besaid to be evidential of the fact that PPARγ plays an important roll inadipocyte differentiation.

Like PPARs, C/EBPs form a family and, recently, it has been found thatC/EBPα, like PPARγ, functions as a master regulator in adipocytedifferentiation. Further, C/EBPβ and C/EBPδ are considered to beexpressed in the early stage of differentiation, controlling theexpression of C/EBPα and PPARγ.

While SREBP1/ADD1 is known to promote the differentiation of adipocytes,it has also been demonstrated that it is involved in PPARγ ligandformation in the process of adipocyte differentiation.

It has been demonstrated that the above-mentioned three transcriptionfactor groups (PPAR, C/EBP, SREBP1/ADD1) make crosstalks, whereby thedifferentiation process goes on.

It has been demonstrated that the expression of the above threetranscription factor groups increases from the relatively early stage ofadipocyte differentiation. They are regarded as master regulatorscontrolling the expression of a plurality of target genes. It is knownthat when these transcription factors are considered from the expressionstage viewpoint, the expression of C/EBPβ and C/EBPδ increases in therelatively early stage. However, what gene or genes are activated in theearliest stage of differentiation of preadipocytes into adipocytes,namely within a half day (12 hours) from the start of differentiation,has been little clarified.

Analyses and/or specification as to whether the genes activated in theearliest stage of differentiation of preadipocytes into adipocytes,namely within 12 hours from the start of differentiation, are expressedor not, the levels thereof, the occurrence or nonoccurrence of proteinsas gene products, the amounts thereof and the sites of expressionthereof, if the genes are expressed, as well as gene mutation analysismay serve to provide factors very important in elucidating themechanisms of obesity, further understanding the progressive state ofobesity properly, and making use of the thus-obtained findings in theprevention or treatment of obesity. For that purpose, it is desirable toidentify such genes as well as the proteins, which are products of suchgenes, and detect or assay such genes or proteins.

Accordingly, it is an object of the present invention to find out a genecapable of being activated within 12 hours from the start ofdifferentiation of preadipocytes into adipocytes and provide a vectorcontaining that gene, a transformant harboring that vector, a proteinproduced from that transformant, an antibody specific to that protein, amethod of producing that protein, an agonist or antagonist to thatprotein, a pharmaceutical composition or diagnostic compositioncontaining one or more of such compounds.

DISCLOSURE OF THE INVENTION

The present inventors made intensive investigations in an attempt toaccomplish the above object and, as a result, they carried out cloningby the subtractive method for the points in time before induction ofadipocyte differentiation and 3 hours after induction using murine3T3-L1 cells (ATCC No. CCL-92.1.) known in the art as a culturedpreadipocyte cell line so that the changes in gene expression at theearliest stage of differentiation might be analyzed. As a result, nearly100 clones were obtained as genes of which the adipocytedifferentiation-promoting protein expression increases within 12 hoursfrom the start of adipocyte differentiation induction and which areactivated within 12 hours from the start of adipocyte differentiationinduction. From among the clones, a polynucleotide having the basesequence shown under SEQ ID NO:1 was obtained as a gene especiallyoutstanding in expression. The base sequence of the polynucleotide asshown under SEQ ID NO:1 was determined using a cDNA expressed in theearly stage of murine adipocyte differentiation, namely before adipocytedifferentiation induction and after 3 hours of induction. Thepolynucleotide is designated as murine clone 24 nucleotide.

Thus, the polynucleotide of the invention is a polynucleotide encoding aprotein having a function to promote adipocyte differentiation andhaving a property such that it is activated within 12 hours from thestart of adipocyte differentiation induction.

In this specification, “activation” means expression or acceleration ofexpression.

Polynucleotides containing a base sequence homologous to the basesequence shown under SEQ ID NO:1, or polynucleotides complementary tosuch polynucleotides were looked up in HYPERLINK“http://www.ncbi.nlm.nih.gov/BLAST/” http://www.ncbi.nlm.nih.gov/BLAST/,whereupon the sequence of a Chinese hamster-derived mRNA for which nofunction is described at all and which has been registered under theaccession number AF371372 with full length of 2794 bp was hit upon. Thehomology between the base sequence shown under SEQ ID NO:1 and the basesequence of the Chinese hamster-derived mRNA is 88.12%, and the homologybetween the portion from the initiation codon to termination codon(bases Nos. 100 to 2520 in SEQ ID NO:1) in base sequence shown under SEQID NO:1 and the known sequence of the Chinese hamster ORF (open readingframe) is 91.72%.

The polynucleotide of the invention is a polynucleotide having a basesequence identical or at least 93%, 95% or 98% homologous to the basesequence shown under SEQ ID NO:1 or a polynucleotide complementary tosaid polynucleotide; it is a polynucleotide which encodes an adipocytedifferentiation-promoting protein capable of being activated within 12hours from the start of adipocyte differentiation induction and isactivated within 12 hours from the start of adipocyte differentiationinduction.

Further, the polynucleotide of the invention is a polynucleotidecontaining a base sequence identical or at least 93%, 95% or 98%homologous to the base sequence from base No. 100 to base No. 2520 ofthe base sequence shown under SEQ ID NO: 1 or a polynucleotidecomplementary to said polynucleotide; it is a polynucleotide encoding aprotein capable of being activated within 12 hours after the start ofadipocyte differentiation induction and having a function to promoteadipocyte differentiation.

A human polynucleotide having a novel base sequence was determined byhuman homolog cloning based on the murine polynucleotide having the basesequence shown under SEQ ID NO:1. The novel base sequence is shown underSEQ ID NO:9.

Thus, the human polynucleotide of the invention is a polynucleotidecontaining the base sequence shown under SEQ ID NO:9 or a polynucleotidecomplementary to said polynucleotide; it is a polynucleotide encoding aprotein having a function to promote adipocyte differentiation andcapable of being activated within 12 hours from the start of adipocytedifferentiation induction; for example, it is a cDNA. Saidpolynucleotide is referred to as human clone 24 nucleotide.

Further, the human polynucleotide of the invention is a polynucleotidecontaining bases Nos. 26 to 2425 of the base sequence shown under SEQ IDNO:9 or a polynucleotide complementary to said polynucleotide; it is apolynucleotide which encodes a protein having a function to promoteadipocyte differentiation and has a property such that it is activatedwithin 12 hours from the start of adipocyte differentiation induction.

The homology between the murine clone 24 polynucloetide shown under SEQID NO:1 and the human clone 24 polynucloetide shown under SEQ ID NO:9 is2089/2444 (mouse vs human), namely 85.47%.

The homology between the sequence of the Chinese hamster-derived mRNAand that of the human polynucleotide is 87.36%, and the homology betweenthe sequence from the initiation codon to termination codon (from baseNo. 26 to base No. 2425) in the human polynucleotide having the basesequence shown under SEQ ID NO:9 and the known sequence of the Chinesehamster ORF is 87.53%.

From the viewpoint of SEQ ID NO:9 and in view of the homology to theknown Chinese hamster ORF sequence (87.36%), the polynucloetide of theinvention includes nucleotides containing a base sequence identical orat least 89%, 91% or 93% homologous to the base sequence shown under SEQID NO:9 or polynucleotides complementary to said polynucleotides.

In view of the homology between the sequence from the initiation codonto termination codon (base No. 26 to base No. 2425 in the base sequence)in the human polynucleotide and the known Chinese hamster ORF sequence(87.53%), the polynucleotide of the invention includes polynucleotidescontaining a base sequence identical or at least 89%, 91% or 93%homologous to the sequence from base No. 26 to base No. 2425 of the basesequence shown under SEQ ID NO:9 or polynucleotides complementary tosaid polynucleotides.

The above-mentioned polynucleotides of the invention can each beextracted from murine cells, human cells or other animal cells within 12hours, in particular at about 6 hours, from the start of adipocytedifferentiation induction. The polynucleotides of the invention can alsobe obtained from genomic DNA, cell/tissue-derived cDNA, acell/tissue-derived cDNA library, or synthetic DNA. The “polynucleotide”of the invention is either DNA or RNA.

The term “clone” simply so referred to herein sometimes means the clone24 nucleotide or clone 24 polynucleotide.

The protein of the invention is a protein capable of being activatedwithin 12 hours, in particular at about 6 hours, from the start ofadipocyte differentiation induction and is a protein promoting adipocytedifferentiation.

The protein of the invention is preferably a protein containing an aminoacid sequence at least 96%, 98% or 100% homologous to the protein aminoacid sequence shown under SEQ ID NO:2 over the full length thereof. Morepreferably, the protein of the invention is the gene translation productof a polynucleotide containing a base sequence at least 96%, 98% or 100%homologous to the base sequence shown under SEQ ID NO:1 over thesequence from base No. 100 to base No. 2520 thereof.

As the polynucleotide containing a base sequence 100% homologous to theamino acid sequence of the protein specified under SEQ ID NO:2, theremay be mentioned a protein capable of being activated in the early stageof murine adipocyte differentiation. This protein is designated asmurine clone 24 protein.

Further, the human protein of the invention includes a proteincontaining the amino acid sequence shown under SEQ ID NO:10. The humanprotein of the invention is the product of gene translation of apolynucleotide containing the sequence from base No. 26 to base No. 2425in the polynucleotide base sequence shown under SEQ ID NO:9. From theSEQ ID NO:10 viewpoint, the invention includes a protein, includingsalts thereof, containing an amino acid sequence at least 93%, 95% or97% homologous to the amino acid sequence shown under SEQ ID NO: 10 overthe full length thereof.

The protein shown under SEQ ID NO:10 is a protein capable of beingactivated within 12 hours, in particular at about 6 hours, from thestart of human adipocyte differentiation induction and is a proteinpromoting adipocyte differentiation. This protein is designated as humanclone 24 protein.

The polynucleotide of the invention is involved in the expression ofPPARγ, C/EBPα, C/EBPδ and SREBP-1, which are proteins expressed afterthe lapse of 12 hours from the start of adipocyte differentiationinduction, and is closely related to the expression of PPARγ, inparticular. Presumably, the protein of the invention directly orindirectly acts on such transcription factors as the PPAR family, C/EBPfamily, and SREBP-1/ADD1. There is the possibility of its stimulatingother pathways as well.

The polynucleotide of the invention or the protein or peptide of theinvention which is produced as a gene translation product of thepolynucleotide is useful in the treatment or diagnosis of alifestyle-related disease selected from among obesity, hypertension,hyperlipidemia, diabetes, and heart diseases or cerebral apoplexyresulting from arteriosclerosis, etc.

Sequence Homology

The reason why the language “identical or at least 93% homologous to abase sequence”, for instance, is used in describing the homology aspectof the invention is that since the homology between the Chinesehamster-derived mRNA registered under the accession number AF371372 witha length of 2794 bp as found in HYPERLINK“http://www.ncbi.nlm.nih.gov/BLAST/” http://www.ncbi.nlm.nih.gov/BLAST/and the base sequence shown under SEQ ID NO:1 is 88.12% and the homologybetween the sequence from the initiation codon to termination codonshown under SEQ ID NO:1 (base No. 100 to base No. 2520 in the basesequence) and the known Chinese hamster ORF sequence is 91.72%, thesequence of said mRNA is to be excluded.

The reason why the language “at least 80% homologous to a base sequenceover the full length thereof” is used in describing another aspect ofthe invention is that the homology on the amino acid level between mouseand human has already been reported to be 84% [Y. Zhang et al., Nature,vol. 372, page 425 (1994); JP-A No. H08-333394]. It has been revealedthat the group of genes involved in lifestyle-related diseases such ashypertension, diabetes and obesity retains 80% or higher homology amonganimals (JP-A No. H11-75865). Therefore, it is reasonable to considerthat as regards the polynucleotide of the invention and the protein ofthe invention as well, the homology among human and other primates andrabbit and other rodents, for instance, will be 80% or higher based onthe sequences disclosed herein. The reasonability of the aboveestimation is supported by the fact that the homology between the murineclone 24 polynucleotide shown under SEQ ID NO:1 and the human clone 24polynucleotide shown under SEQ ID NO:9 is 85.47%.

Cloning of the Polynucleotide of the Invention

As for the means of cloning a polynucleotide encoding the protein of theinvention, it can be amplified by the PCR method using synthetic DNAprimers each having a part of the base sequence of the protein of theinvention, or it can be selected by hybridization using labeled DNAfragment or synthetic DNA encoding a part or the whole region of theprotein of the invention against an appropriate vector containing DNAinsert, for instance. The hybridization can be carried out by the methoddescribed in Molecular Cloning, 2nd edition (J. Sambrook et al., ColdSpring Harbor Lab. Press, 1989), for instance. In cases where acommercial library is used, it can be carried out according to theprocedure described in the manual attached thereto. According to theintended purpose thereof, the cloned protein-encoding DNA can be usedeither as such or, after digestion with a restriction enzyme wheredesired or after addition of a linker. The DNA may have ATG as thetranslation initiation codon on the 5′ terminal end and may have TAA,TGA or TAG as the translation termination codon on the 3′ terminal end.It is also possible to add these translation initiation codon andtranslation termination codon using appropriate synthetic DNA adaptors.

Vector etc. for the Expression of the Polynucleotide

The vector of the invention is a recombinant vector containing apolynucleotide identical or at least 93% homologous to thepolynucleotide shown under SEQ ID NO:1 or a polynucleotide complementaryto said polynucleotide and preferably is a recombinant vector containinga polynucleotide containing the base sequence from base No. 100 to baseNo. 2520 in the base sequence shown under SEQ ID NO:1.

Further, the vector of the invention which contains a polynucleotideinvolved in human adipocyte differentiation is a recombinant vectorcontaining a polynucleotide containing the base sequence shown under SEQID NO:9 or a polynucleotide complementary to said polynucleotide andpreferably is a recombinant vector containing a polynucleotidecontaining the base sequence from base No. 26 to base No. 2425 in thebase sequence shown under SEQ ID NO:9 or a polynucleotide complementaryto said polynucleotide.

The vector having the polynucleotide sequence of the invention can beconstructed in the conventional manner. The vector suited for suchpurpose has a promoter region upstream of the insertion site of thepolynucleotide of the invention. This promoter may be a known one andcan be selected according to the host cell. In cases where Escherichiacoli or a like bacterial species is used as the host, the lac promoter,trp promoter, T7 promoter, tac promoter, or λPL promoter, for instance,can be utilized. When a yeast is used as the host, the GADPH promoter,ADH promoter, PGK promoter, and PH05 promoter can be utilzed. Whenanimal-derived cells are used as the host, there may be mentioned thehuman cytomegalovirus promoter, SV40 virus-derived promoter, EF-1 αpromoter, β actin promoter, and metallothionein promoter. Preferably,the vector for the expression of the polynucleotide according to theinvention has a transcription termination signal downstream from thesite of insertion of the polynucleotide of the invention. Furthermore,it is desirable that the vector contains a marker for identification,for example a drug resistance marker.

Host

As for the host that can be used in the practice of the invention,bacteria of the genus Escherichia, bacteria of the genus Bacillus,yeasts, insect cells, insects, animal cells and so forth are suitable.

As typical examples of the bacteria of the genus Escherichia,Escherichia coli JM109 [ATCC 53323, product of Toyobo Co., Ltd.], JM103[Nucleic Acids Research, vol. 9, 309 (1981), K12•DH1 [Proc. Natl. Acad.Sci. USA, vol. 60, 160 (1968), JA221 [Journal of Molecular Biology, vol.120, 517 (1978)], HB11 [Journal of Molecular Biology, vol. 41, 459(1969)], and C600 [Genetics, vol. 39, 440 (1954)], among others arementioned.

As the bacteria of the genus Bacillus, among others, Bacillus subtilisM1114 [Gene, vol. 24, 255 (1983)], and 207-21 [Journal of Biochemistry,vol. 95, 87 (1984)] are mentioned.

As the yeasts, Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D,20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, and Saccharomycespichiapastoris, among others are mentioned.

As for the insect cells, when the virus is AcNPV, cabbage armywormlarva-derived established cell line cells (Spodoptera frugiperda cells;Sf cells), Trichoplusia ni mesenteron-derived MG1 cells, Trichoplusia niegg-derived High Five™ cells, Mamestra brassicae-derived cells, andEstigmena acrea-derived cells are mentioned. When the virus is BmNPV,silkworm-derived established cell line cells (Bombyx mori N cells; BmNcells), among others are mentioned. As the above Sf cells, for example,Sf9 cells (ATCC CRL1711), and Sf21 cells (for both, Vaughn, J. L. etal., In Vivo, vol. 13, 213-217 (1977)) are mentioned.

As the insects, silkworm larvae [Maeda et al., Nature, vol. 315, 592(1985)] is mentioned, for instance.

Usable as the animal cells are, for example, monkey COS-7, Vero, Chinesehamster cells CHO (hereinafter referred to as CHO cells for short), dhfrgene-deficient Chinese hamster cells CHO [abbreviation: CHO(dhfr−)cells], mouse L cells, mouse AtT-20, murine myeloma cells, rat GH3,human FL cells.

Transformant Production

The transformant of the invention is derived from the above-mentionedhost by allowing it to harbor the recombinant vector mentioned above.

Bacteria of the genus Escherichia can be transformed according to themethod described in Proc. Natl. Acad. Sci. USA, vol. 69, 2110 (1972), orGene, vol. 17, 107 (1982), for instance.

Bacteria of the genus Bacillus can be transformed according to themethod described in Molecular and General Genetics, vol. 168, 111(1979), for instance.

Yeasts can be transformed according to the method described in Methodsin Enzymology, vol. 194, 182-187 (1991), or Proc. Natl. Acad. Sci. USA,vol. 75, 1929 (1978), for instance.

Insect cells or insects can be transformed according to the methoddescribed in Bio/Technology, vol. 6, 47-55 (1988), for instance.

Animal cells can be transformed according to the method described inSaibo Kogaku (Cell Engineering), Supplement No. 8, Saibo Kogaku JikkenPurotokoru (Protocols for Cell Engineering Experiments), 263-267 (1995)(published by Shujunsha), or Virology, vol. 52, 456 (1973), forinstance.

Transformant Cultivation

The transformant of the invention can be cultured under conditionssufficient for protein formation with the conditions mentioned belowbeing taken into consideration and, as a result, the desired protein canbe recovered from the culture medium or transformant.

When the host is a bacterial strain of the genus Escherichia orBacillus, the transformant is preferably cultured in a liquid medium,which contains carbon sources, nitrogen sources, inorganic substancesand other factors necessary for the growth of the transformant. As thecarbon sources, there may be mentioned glucose, dextrin, soluble starch,and sucrose, among others. As the nitrogen sources, there may bementioned ammonium salts, nitrate salts, corn steep liquor, peptone,casein, meat extracts, soybean meal, potato extracts and like inorganicor organic substances. As the inorganic substances, there may bementioned calcium chloride, sodium dihydrogen phosphate, and magnesiumchloride, among others. Yeasts, vitamins, growth promoting factors andthe like may be added to the medium. The medium desirably has a pH ofabout 5 to 8.

In culturing bacteria of the genus Escherichia, M9 medium containingglucose and casamino acids (Miller, Journal of Experiments in MolecularGenetics, 431-433, Cold Spring Harbor Laboratory, New York, 1972) ispreferably used. If necessary, such an agent as 3-indolylacrylic acid,for instance, may be added thereto for effective promoter functioning.In cases where the host is a bacterial strain of the genus Escherichia,the cultivation is generally carried out at about 15-43° C. for about3-24 hours. If necessary, aeration and/or agitation can be made.

In cases where the host is a bacterial strain of the genus Bacillus, thecultivation is generally carried out at about 30-40° C. for about 6 to24 hours. If necessary, aeration and/or agitation can be performed. Whenthe host is a yeast, the transformant is cultured, for example, inBurkholder minimal medium [Bostian, K. L. et al., Proc. Natl. Acad. Sci.USA, vol. 77, 4505 (1980)] or SD medium containing 0.5% of casaminoacids [Bitter, G. A. et al., Proc. Natl. Acad. Sci. USA, vol. 81, 5330(1984)]. The medium is preferably adjusted to a pH of about 5 to 8. Thecultivation is generally carried out at about 20° C.-35° C. for about24-72 hours, if necessary with aeration and/or agitation.

In culturing a transformant derived from an insect cell host or insecthost, Grace's insect medium (Grace, T. C. C., Nature, vol. 195, 788(1962)) adequately supplemented with 10% inactivated bovine serum and/orsome other additive or additives is used as the medium, among others.The pH of the medium is preferably adjusted to about 6.2 to 6.4. Thecultivation is generally carried out at about 27° C. for about 3-5 days,if necessary with aeration and/or agitation.

In culturing a transformant derived from an animal cell host, MEM medium[Science, vol. 122, 501 (1952)], DMEM medium [Virology, vol. 8, 396(1959)], RPMI 1640 medium [Journal of the American Medical Association,vol. 199, 519 (1967)], or 199 medium [Proceedings of the Society for theBiological Medicine, vol. 73, 1 (1950)] containing about 5-20% of fetalbovine serum, for instance, is used as the medium. The pH is preferablyabout 6 to 8. The cultivation is generally carried out at about 30°C.-40° C. for about 15-60 hours, if necessary with aeration and/oragitation. In the above manner, the protein of the invention can beformed in the transformant cells.

Protein Formation in a Cell-Free System

In addition to the protein production by transformant cultivation asmentioned above, it is also possible to form the desired protein using acell-free cultivation system such as a high-efficiency cell-free proteinsynthesis system of Invitrotech (HYPERLINK“http://www.invitrotech.co.jp/”) or Roche's in vitrotranslation/transcription system, for instance.

Protein Isolation and Purification

The protein of the invention can be isolated and purified, for example,by the method mentioned below. In extracting the protein of theinvention from cultured bacterial cells or other cells, a methodcomprising collecting bacterial cells or other cells after culturing inthe conventional manner, suspending them in an appropriate buffer,disrupting them by sonication, lysozyme treatment and/or freezing andthawing, and recovering a crude protein-containing extract bycentrifugation or filtration, for instance, or a similar method isadequately used. The buffer solution may contain a protein denaturingagent, such as urea or guanidine hydrochloride, or a detergent such asTriton X-100 (trademark, product of Union Carbide). Usable as such knownmethod of isolation/purification are such solubility-based methods assalting out and solvent precipitation, such methods mainly based onmolecular weight differences as dialysis, ultrafiltration, gelfiltration and SDS-polyacrylamide gel electrophoresis, such chargedifference-based methods as ion exchange chromatography, such specificaffinity-based methods as affinity chromatography, such hydrophobicitydifference-based methods as reversed phase high performance liquidchromatography, and such isoelectric point difference-based methods asisoelectric focusing, among others.

In cases where the protein obtained by such a method as mentioned aboveis in a free form, it can be converted to a salt form by a known methodor a method based thereon. Conversely, when it is obtained in a saltform, it can be converted to a free form or another salt form by a knownmethod or a method based thereon. It is also possible to arbitrarilymodify the protein produced by the recombinant or partially deprive apolypeptide from the protein by causing an appropriate protein-modifyingenzyme to act thereon before purification or after purification. Usableas the protein-modifying enzyme are, for example, trypsin, chymotrypsin,arginine endopeptidase, protein kinase, and glycosidase. The presence ofthe thus-formed protein of the invention or a salt thereof can beconfirmed by a labeled ligand binding assay or an enzyme immunoassayusing a specific antibody.

Human Homolog Cloning

It is possible to find out a highly homologous sequence and predict thefull length of the human homolog by homology search against NCBI GenomeSequencing (Human Genome Database), using the murine full-length DNAsequence shown under SEQ ID NO: 1 as determined in the manner mentionedabove, which is a polynucleotide capable of being activated within 12hours from the start of adipocyte differentiation induction. The RT-PCRmethod, for instance, can be applied to human homolog cloning. The fulllength of the human homolog predicted as above and actually sequenced bythe RT-PCR method is the base sequence shown under SEQ ID NO:9. From thefull length base sequence of the human homolog as shown under SEQ IDNO:9, it can be predicted that said base sequence is a gene encoding aprotein composed of 800 amino acid residues and that the amino acidsequence of said protein is as shown under SEQ ID NO:10.

Method of Antagonist and Agonist Identification

The method of identifying a compound antagonizing or agonizing theprotein of the invention comprises the following steps (a) and (b).

-   (a) The step of bringing a candidate compound into contact with    cells in which the protein of the invention is being expressed or    cells responsive to the protein of the invention;-   (b) The step of observing as to binding, or stimulation or    inhibition as a functional response; or the step of comparing the    cells that have been contacted with the candidate compound with the    cells of the same kind that have not been contacted with the    candidate compound with respect to an ability associated with an    activity of the protein of the invention.

The antagonist or agonist of the invention can be identified by theabove method of identification.

Method of Polynucleotide Detection; Diagnosis Based on thePolynucleotide

A disease in a subject to be diagnosed which is associated with theactivation of the protein of the invention in the subject, or thesensitivity to such disease can be diagnosed in the following manner.The diagnosis is performed by determining as to whether there is amutation in the base sequence encoding the protein of the invention inthe genome of the subject to be diagnosed or not; and/or analyzing theoccurrence or amount of the protein of the invention in a sample derivedfrom the subject.

For detecting or assaying the polynucleotide of the invention, primerscomprising not less than 8 consecutive bases but not more than 100consecutive bases selected from the base sequence of the polynucleotideof the invention are synthesized and, separately, mRNA is extracted fromcells or blood, for instance, and amplified by the RT-PCR method whileconverting mRNA to DNA, or the target polynucleotide is amplified by theT7-based mRNA amplification method (Van Gelder, R. N. et al., Proc.Natl. Acad. Sci. USA, 87: 1663-1667, 1990). The thus-amplifiedpolynucleotide can be detected by any of various electrophoretictechniques. It is also possible to add Cy3-dUTP or Cy5-dUTP in the stepof polynucleotide amplification for fluorescent labeling of theamplified polynucleotide and detect the labeled polynucleotide using aDNA microarray (Brown, P. O. et al.: Nature Genet., 21, sup.: 33-37,1999).

For assaying the mRNA extracted, the quantitative PCR method using afluorescent dye is carried out [Yodosha (publisher): non-R1-Jikken noSaishin Purotokoru (Latest Protocols for non-R1 Experiments), edited byKurihara et al., 1999, pp. 83-89]. This method reveals the extent ofoccurrence of the mRNA in cells. Based on that extent of expressionthereof, the condition of obesity can be checked.

It is also possible to detect and measure a plurality of polynucleotidessimultaneously using a DNA microarray. For example, by reacting thelabeled polynucleotide prepared from a sample with a plurality ofpolynucleotides including the polynucleotide of the invention as boundonto a substrate, it becomes possible, in the manner mentioned above, tojudge which polynucleotide on the substrate is reactive with the labeledpolynucleotide and as to whether there is any reactive polynucleotide ornot. This judgment makes it possible to understand the condition of someor other lifestyle-related disease and utilize the findings in thetreatment thereof. Further, by allowing a plurality of obesity-relatedpolynucleotides, including of the polynucleotide of the invention, to bebound to a substrate, it becomes possible to understand the conditionand extent of obesity by the same technique and utilize the findings inthe treatment thereof.

As regards the detection of the polynucleotide of the invention, it isalso possible to detect the same by capillary electrophoresis utilizingApplied Biosystem's ABI PRISM 310, PRISM 3100 or PRISM 3700.

It has been revealed that single nucleotide polymorphisms (hereinafter,SNPs) resulting from substitution of some or other base for a specificnucleic acid base in a polynucleotide and resulting in modification inactivity of a protein as a gene product, modification in the ability ofthe protein of the invention to bind to a receptor thereof, modificationin reactivity of said protein with a drug, modification in the stabilityof said protein, or suspension of the synthesis of said protein, arevery important. As for the polynucleotide of the invention as well, thesignificance of detecting such SNPs is very important.

If a high-density SNP marker gene map for the adipocytedifferentiation-related polynucleotides of the invention becomeavailable, might make it easy to make comparisons between healthysubjects and patients based on SNPs that can specify the genes causativeof diabetes. If such a high-density SNP map is used, it will becomepossible to perform a correlation analysis (whole genome associationstudy) using a large size of non-family-related samples. Throughsubstitution of one base, in particular, SNPs result in changes in thecorresponding amino acid, which changes in turn lead to changes inphysical properties of the protein proper. On the enzyme level, forinstance, a decrease or increase in enzyme activity as resulting fromthe change of an amino acid in the vicinity of the active center and, onthe receptor level, a decrease or increase in binding ability asresulting from the change of an amino acid in the vicinity of theacceptor center. The adjustment of the dose of a drug and/or theselection of a different drug according to the gene polymorphism of eachindividual subject is referred to tailored medicine, for instance.

Applicable as the method of detecting SNPs in the polynucleotides of theinvention are, for example, the method of using U.S. Nanogen's(http://www.nanogen.com) nanochips (Gilles et al., Nature Biotechnology,365-370, 17, 1999), the method comprising polynucleotide sequencedetermination (HYPERLINK “http://www.pyrosequencing.com/”http://www.pyrosequencing.com), the method using DNA chips or DNAarrays, the method using a mass spectrometer (Legler et al.,Transfusion, 36: 426-431, 1996), the method using primer extensions, theLuminex method (Iannone et al., Cytometry, 39: 131-140, 2000), etc.

As the diagnostic agent to be constituted for detecting each of thepolynucleotides mentioned above, there may be mentioned a diagnosticagent comprising a polynucleotide having a sequence comprising at least10 consecutive bases taken out of those polynucleotides containing abase sequence identical or at least 80% homologous to the base sequenceshown under SEQ ID NO:1 or those polynucleotides complementary to saidpolynucleotides.

Further, the diagnostic agent may comprise a polynucleotide having asequence comprising at least 10 consecutive bases taken out of thosepolynucleotides containing a base sequence identical or at least 80%homologous to the sequence from base No. 100 to base No 2520 in the basesequence shown under SEQ ID NO:1 or those polynucleotides complementaryto said polynucleotides. The diagnostic agent constituted for humanpolynucleotide detection, in particular, is a diagnostic agentcomprising a polynucleotide having a base sequence comprising at least10 consecutive bases taken out of those polynucleotides containing thebase sequence shown under SEQ ID NO:9 or those polynucleotidescomplementary to said polynucleotides.

More preferably, the diagnostic agent constituted for humanpolynucleotide detection is a diagnostic agent comprising apolynucleotide having a sequence comprising at least 10 consecutivebases taken out of those polynucleotides containing the base sequencefrom base No. 26 to base No. 2425 in the base sequence shown under SEQID NO:9 or those polynucleotides complementary to said polynucleotides.

Detection of the Protein or Peptide, Detection of an Antibody to theProtein or Peptide, and Application to Diagnosis

For detecting the protein of the invention, the immunoassay method isgenerally used. For example, mice, rats, rabbits, goats, swine, cows andbulls, sheep, chickens or like animals can be immunized, for antibodyproduction, with the protein shown under SEQ ID NO:2 or SEQ ID NO:10 ora peptide having an amino acid sequence composed of three or moreconsecutive amino acid residues as contained in either of the sequences.In carrying out this immunization, it is a general practice to add asubstance called adjuvant. Usable as the adjuvant are Freund's completeadjuvant, Freund's incomplete adjuvant, alum, and various other knownadjuvants. In the case of immunizing mice, the spleen of each immunizedmouse is excised after a certain period of immunization, and thesplenocytes, which are antibody-producing cells, are fused to myelomacells, and hybridomas producing an antibody specifically binding to theprotein shown under SEQ ID NO:2 are prepared by carrying out cloning.

An antibody produced upon cultivation of such a hybridoma is purified byvarious methods, and the antibody obtained is immobilized on a solidphase. Separately, the antibody is labeled with an enzyme, fluorescentdye, metal colloid, latex, DNA or RNA, for instance, to give a labeledantibody. The above immobilized antibody and labeled antibody arereacted with a sample such as cells, blood or one of the fractionsthereof, and the presence or absence or the amount of the label bound tothe solid phase is checked or assayed, whereby the protein of theinvention in the sample can be quantitated.

Since the protein of the invention is specifically found in adipocytes,the quantitation thereof makes it possible to understand the extent andcondition of obesity.

For assaying the protein of the invention contained in samples, anantibody is prepared by immunizing rabbits, rats, sheep or goats withthat protein or a partial peptide of that protein as an antigen (in thiscase, the preparation of a monoclonal antibody capable of binding to aspecific recognition site being included). The thus-obtained antibody isallowed to be physically adsorbed on such a material as polystyrene, alatex or nitrocellulose, or biotin is introduced into the antibody inadvance and the biotinylated antibody is reacted with streptavidin,avidin or the like bound in advance to a solid phase for the preparationof a solid phase, or an antibody-bound solid phase is prepared bycovalently binding the antibody to a solid phase via a carboxyl, amino,sulfhydryl or like group occurring on the solid phase. The thus-obtainedantibody-bound solid phase is reacted with a sample, such as cells,blood or any of the fractions thereof for binding the antigen containedin the sample to the solid phase and, then, the antigen bound to theantibody-bound solid phase is reacted with a labeled antibody derivedfrom the antibody prepared by the above-mentioned process by bindingthereto a radioisotope, enzyme, fluorescent dye or nucleic acid orbiotin or a like label. The steps of reacting the antibody-bound solidphase with the sample and with the labeled antibody can also be carriedout simultaneously. By assaying the label thus bound to the solid phase,it is possible to assay the protein of investigation contained in thesample.

A different method such as mentioned below can also be used as themethod of antibody detection. Thus, the protein of the invention or apartial peptide of that protein is used as an antigen and immobilized ona solid phase to give an antigen-bound solid phase. Then, theantigen-bound solid phase is reacted with a substance obtained bylabeling the antigen and a sample such as cells, blood or any of thefractions thereof, and the label bound is quantitated, whereby theantibody amount contained in the sample can be assayed. Therefore, adiagnostic agent capable of detecting an antibody immunologicallyspecific to the protein of the invention can be constructed.

While the reaction systems described above all comprises the antigen orantibody directly immobilized on a solid phase, a reaction systemcomprising an oligonucleotide bound to a solid phase and an antigen orantibody bound to an oligonucleotide complementary to thatoligonucleotide as bound to the solid phase, as disclosed in U.S. Pat.No. 5,789,165, is also applicable. Furthermore, as described in JP-A No.H10-253632 or EP 0905517 A1, it also becomes possible to detect or assaydifferent items simultaneously. For example, it presumably becomespossible to simultaneously detect markers associated withlifestyle-related diseases such as diabetes, hypertension and obesityand judge the level of such lifestyle-related diseases depending on thedegrees of detection of respective proteins. Further, it presumablybecomes possible to detect and assay such markers as obesity-relatedproteins or peptides simultaneously, and understand the state of obesityand utilize the findings in the treatment of obesity.

For immunologically detecting the protein of the invention or anantibody to that protein, there may be mentioned, in addition to themethods described above, the method involving immunoagglutination (JP-ANo. H06-3358), the surface plasmon resonance (SPR) method using BIAcoreof Biacore International AB (Sweden) (http:/www.biacore.co.jp), forinstance, and the method using a crystal oscillator (JP-A No.1109-292397), among others.

As the diagnostic agent constituted for detecting the above-mentionedprotein of the invention, there may be mentioned a diagnostic agentcomprising the protein of the invention or a salt thereof. In apreferred embodiment, the diagnostic agent of the invention is adiagnostic agent comprising a protein or peptide comprising at least 5consecutive amino acid residues out of the protein of the invention or asalt thereof. Another diagnostic agent for detecting the protein of theinvention comprises an antibody to the protein of the invention.

Use for Therapeutic Purposes

The present invention provides a method of therapeutic treatment ofthose abnormal conditions such as lifestyle-related diseases, includingobesity, hypertension, hyperlipidemia, diabetes, renal diseases, insulintolerance, lipodystrophy, CNS diseases, and/or heart diseases orcerebral apoplexy resulting from arteriosclerosis, which are related tothe activity of an excessive or insufficient amount of the protein ofthe invention or a peptide constituting a part of that protein. In caseswhere the activity of the protein of the invention or a peptideconstituting a part of that protein is excessive, several methods can beused.

One measure is characterized in that a pharmaceutical compositioncomprising an effective amount of the above-mentioned inhibitor compound(antagonist) in combination with a pharmaceutically acceptable carrieris administered to a subject to be treated to thereby block the ligandbinding to the protein or peptide of the invention or inhibit the secondsignal and inhibit the activation so that the abnormal condition may beimproved.

Another measure is characterized in that a pharmaceutical compositioncomprising a therapeutically effective amount of a compound capable ofactivating the polynucleotide of the invention (i.e. agonist) incombination with a pharmaceutically acceptable carrier is administeredso that the abnormal condition may be improved.

In another approach, the protein or peptide of the invention or a saltthereof in a soluble form, which is capable of binding to a ligand incompetition with the protein or peptide of the invention, may beadministered. Typical examples of such competitor substance include theprotein of the invention and peptides constituting a part of thatprotein. Thus, the pharmaceutical composition of the invention may be apharmaceutical composition comprising said protein or a salt thereof, ora peptide composed of at least 5 consecutive amino acid residues of saidprotein or a salt thereof.

According to a further technique, which utilizes the expression blockingmethod, the polynucleotide of the invention or a polynucleotidecomplementary to that polynucleotide may be administered to inhibit thegene activation.

Such pharmaceutical composition of the invention is a pharmaceuticalcomposition comprising a polynucleotide having a base sequence identicalor at least 80% homologous to the base sequence of the clone 24polynucleotide as shown under SEQ ID NO:1, or a polynucleotidecomplementary to that polynucleotide.

Preferably, it is a pharmaceutical composition comprising apolynucleotide having a base sequence comprising at least 10 consecutivebases taken out of those polynucleotides containing a base sequenceidentical or at least 80% homologous to the base sequence shown underSEQ ID NO:1 or those polynucleotides complementary to saidpolynucleotides and, more preferably, it is a pharmaceutical compositioncomprising a polynucleotide having a base sequence comprising at least10 consecutive bases taken out of those polynucleotides containing abase sequence identical or at least 80% homologous to the sequence frombase No. 100 to base No. 2520 in the base sequence shown under SEQ IDNO:1 or to those polynucleotides complementary to said polynucleotides.

As regards the human clone 24 polynucleotide, in particular,polynucleotides containing the base sequence shown under SEQ ID NO:9 orpolynucleotides complementary to those polynucleotides are taken intoconsideration. Preferred is a pharmaceutical composition comprising apolynucleotide having a base sequence comprising at least 10 consecutivebases taken out of those polynucleotides containing the base sequenceshown under SEQ ID NO:9 or those polynucleotides complementary to saidpolynucleotides. More preferred is a pharmaceutical compositioncomprising a polynucleotide having a base sequence comprising at least10 consecutive bases taken out of those polynucleotides containing thesequence from base No. 26 to base No. 2425 in the base sequence shownunder SEQ ID NO:9 or to those polynucleotides complementary to saidpolynucleotides.

These pharmaceutical compositions utilize, as a therapeutic agent, anantisense to a sequence around ATG. In these pharmaceuticalcompositions, an antisense sequence formed within cells or separatelyadministered can be utilized in the conventional manner. Refer, forexample, to Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988), and O'Connor, J.Neurochem. (1991) 56: 560. As an alternative, a triple helix-formingoligonucleotide may be provided together with the gene. Cf. e.g. Lee etal., Nucleic Acids Res. (1979) 6: 3073; Cooney et al., Science (1988)241: 456; Dervan et al., Science (1991) 251: 1360. These oligomersthemselves may be administered as such, or related oligomers may beactivated invivo.

As an alternative, the formation of the polynucleotide of the inventionin cells of a subject to be treated may be made effective by means ofgene therapy. For example, the polynucleotide of the invention may betreated, as mentioned above, and inserted into a replicative defectiveretrovirus vector for activation thereof. Then, the retrovirusexpression construct is isolated and introduced into packaging cellstransduced with a retrovirus plasmid vector containing the RNA encodingthe protein of the invention and, then, the packaging cells are allowedto form infective viral particles containing the desired polynucleotide.These producer cells may be administered to a subject to be treated forinvivo treatment of the cells and invivo activation of the protein.Applicable to the gene therapy are the methods described in HumanMolecular Genetics, T. Strachan and A. P. Read, BIOS ScientificPublishers Ltd. (1986), Chapter 20, Gene Therapy and other MolecularGenetic-based Therapeutic Approaches (and references cited therein).

A pharmaceutical composition can be prepared by incorporating anantibody to the clone 24 protein or human clone 24 protein of theinvention or to a peptide comprising a part of the amino acid sequenceconstituting either of the proteins.

In cases where such antibody is administered, it can be expected thatthe adipocyte differentiation activity may be reduced.

Formulation and Administration

In the treatment of a subject to be treated who requires enhancedactivation of the protein of the invention, a therapeutically effectiveamount of a protein containing an amino acid sequence identical or atleast 80% homologous to the whole length of the protein of the inventionas shown under SEQ ID NO:2, or a peptide constituting a part of thatprotein, or a salt thereof, an antibody to the protein or a peptideconstituting a part of the protein, an agonist, an antagonist peptide,or a size-reduced molecule derived therefrom, each in a soluble form,may be administered to a subject to be treated.

Such formulation comprises a therapeutically effective amount of theabove-mentioned substance or a salt thereof, and a pharmaceuticallyacceptable carrier or excipient. Such carrier includes, but is notlimited to, saline, buffered saline, dextrose, water, glycerol, ethanol,and mixtures of these. The formulation should be adequate for the routeof administration, as is well known to those skilled in the art. Thepresent invention is further concerned with a pharmaceutical pack or kitcomprising one or more containers with one or more of theabove-mentioned active ingredients of the invention contained therein.

The protein and other compounds of the invention may be used singly orin combination with some other compounds such as therapeutic compounds.A preferred form of systemic administration of the pharmaceuticalcomposition includes injection, typically intravenous injection. Otherroutes of injection, such as hypodermic, intramuscular orintraperitoneal, may also be used. Another means for systemicadministration includes administration through a mucous membrane orpercutaneous administration using a penetrant such as a bile acid saltor fusidic acid or some other surfactant. If a satisfactory formulationwhich melts in the entrails or capsule formulation is available, oraladministration is also possible. These compounds may be administeredlocally, and they may be in the form of plasters or ointments, pastes,gels, etc.

The necessary dose range depends on the peptide, route ofadministration, nature of formulation, nature of symptom of the subjectto be treated, and judgment of the doctor in charge. An appropriate doseis within the range of 0.1 to 100 μg per kilogram of body weight of thesubject to be treated. Considering the differences in efficacy among thevarious compounds to be used and among various routes of administration,however, the necessary dose range is estimated to be wide. For example,higher doses are estimably required in oral administration than inintravenous injection. The doses can be varied using standard androutine experiments for optimization, as well understood in the relevantfield of art.

In the above-mentioned method of treatment, which is frequently referredto as “gene therapy”, it is also possible to cause the protein of theinvention, which is to be used for the treatment, to be formed in asubject to be treated. Therefore, for example, cells derived from thesubject to be treated may be treated exvivo using a retrovirus plasmidvector and a polynucleotide such as a protein-encoding DNA or RNA. Thecells treated are then introduced into the subject to be treated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of the clone 24 mouse cDNA andfurther illustrates the subcloning for base sequence determination ofthe clone 24 based on the information about the DNA fragment Su obtainedby the subtraction technique.

FIG. 2 is an autoradiogram showing that a labeled probe prepared fromthe clone 24 reacts with mRNA samples prepared from cells from 3 hoursto 12 hours after the start of differentiation induction, with a peak at6 hours after the start of differentiation induction.

FIG. 3(c) is a photomicrograph of the GFP of the clone 24 expressed in acell. FIG. 3(a) is a photomicrograph of the whole cell as a control.FIG. 3(b) is a photomicrograph of the nucleus in the cytoplasm asstained by the DAPI method.

FIG. 4 is a schematic representation of the clone 24 polypeptide. In thesequence, the sequence site considered to be a leucine zipper structureis shown in detail.

FIG. 5 shows an autoradiogram of NIH/3T3 cells showing ectopicexpression of the clone 24.

FIG. 6 schematically shows the clone 24 human homolog cDNA.

FIG. 7 shows the results of Oil-Red-O staining of NIH/3T3 cells showingectopic expression of the clone 24 on day 8 after differentiationinduction and of control cells.

FIG. 8 is a graphic representation of the results of identifyingantibodies to the clone 24, which results indicate that the antibodiesobtained by immunization with PEPTIDE 4, PEPTIDE 8 and PEPTIDE 10, whichare partial peptides constituting the clone 24 protein, react with asolid module carrying the respective peptides immobilized thereon.

EXAMPLE 1

Confirmation of Differentiation into Adipocytes

Murine 3T3-L1 cells (ATCC No. CCL-92.1.) capable of differentiating intoadipocytes were cultured in a collagen type I dish (product of FALCON)using a basal medium (DMEM, 4 μg/ml KM, 10% calf serum) in 5% CO₂ at 37°C. After the lapse of 2 days after arrival of cells at a confluentstate, when cells were in the resting phase, the medium was replacedwith a differentiation inducing medium (DMEM, 40 μg/ml KM, 10% FBS, 0.5mM 1-methyl-3-isobutylxanthine (Mix), 10 μg/ml insulin, 1 μMdexamethasone (Dex)), cultivation was carried out under thesedifferentiation conditions for 48 hours, and the medium was replacedwith a differentiation promoting medium (DMEM, 40 μg/ml KM, 10% FBS, 5μg/ml insulin). Medium change was carried out with the differentiationpromoting medium at 2-day intervals. It was confirmed that murine 3T3-L1cells (ATCC No. CCL-92.1) began to contain small fat drops at around the4th day and thereon and, after one week, had differentiated into matureadipocytes.

EXAMPLE 2

Preparation of mRNA

Using TRIzol (product of GIBCO BRL) and according to the manual attachedto TRIzol (product of GIBCO BRL), the whole amount of RNA of the murine3T3-L1 cell origin was prepared from murine 3T3-L1 cells (ATCC No.CCL-92.1.) before adipocyte differentiation induction or at 3 hoursafter the start of differentiation induction. mRNA before adipocytedifferentiation induction and mRNA at 3 hours after adipocytedifferentiation induction were prepared from the respective wholeamounts of RNA using Oligotex-dT 30 (product of Daiichi Pure Chemicals).

EXAMPLE 3

Activity Measurement at 3 Hours from Adipocyte Differentiation Inductionby the PCR-Select cDNA Subtraction Method

The following steps i) to vii) were carried out using the known PCRselect cDNA subtraction kit (product of CLONTECH Laboratories).

i) Synthesis of Tester cDNA and Driver cDNA

The murine 3T3-L1 cell (ATCC No. CCL-92.1.)-derived mRNA beforeadipocyte differentiation induction as prepared in Example 2 was used asthe driver mRNA, and the mRNA prepared in Example 2 at 3 hours afteradipocyte differentiation induction was used as the tester mRNA.Single-stranded cDNAs were synthesized from the driver mRNA and testermRNA and, then, a double-stranded driver cDNA and a double-strandedtester cDNA were synthesized.

ii) Double-Stranded cDNA Cleavage with the Restriction Enzyme RsaI

The double-stranded driver cDNA and tester cDNA obtained in the abovestep i) were each cleaved with the restriction enzyme RsaI, andblunt-ended fragments were prepared.

iii) Adapter Ligation

The tester cDNA obtained in the above step ii) was divided into twogroups. Adaptor 1 was ligated to one group, and adaptor 2 was ligated tothe other group.

iv) First Hybridization

The two kinds of the tester cDNAs obtained by adapter ligation in theabove step iii) were separately subjected to hybridization with anexcessive amount of the driver cDNA.

v) Second Hybridization

Further, an excessive amount of the driver cDNA was mixed with the twokinds of the tester cDNAs resulting from hybridization with the drivercDNA as obtained in the above step iv), and hybridization was carriedout.

vi) First PCR

The hybridized tester cDNA obtained in the above step iv) was amplifiedby the PCR method using a primer common to the two adapter sites.

vii) Second PCR

Finally, the DNA was amplified using two primers specific to the twoadapter sites.

The tester cDNA hybridized with the driver cDNA was not amplified. Onthe other hand, the tester-specific polynucleotide (gene), namely thepolynucleotide showing an increased degree of activation after 3 hoursof differentiation induction, alone was amplified.

EXAMPLE 4

PCR Product Subcloning and Clone 24 Plasmid

The clone 24 murine cDNA is schematically shown in FIG. 1. The amplifiedpolynucleotide-containing reaction mixture after carrying out the secondPCR as obtained by the subtraction method in Example 3 was subjected tophenol extraction, followed by CIAA extraction and EtOH precipitation.The DNA pellet obtained was dissolved in 17 μl of sterilized water, andadapter cleavage was effected using 10 units of RsaI at 37° C. overnightunder the conditions: 10 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT.After 0.7% agarose gel electrophoresis and purification using DB81(product of Whatman), the DNA fragment Su (cf. FIG. 1) was obtained.

Based on the information about the DNA fragment Su, primers weredesigned, and 5′-RACE was carried out. As a result, a band of about 2Kbp was obtained. This was recovered and designated as nucleotide R-5′(cf. FIG. 1). The nucleotide R-5′ was subcloned in T vector (pBluescriptKS+) and sequenced using DSQ-1000 (product of Shimadzu Corp.). On theother hand, data base retrieval using the 3′ side sequence of the clone24 revealed an identical base sequence registered in EST and, referringto this base sequence, primers were designed and RT-PCR was carried out.As a result, a band of about 700 bp was obtained. This was recovered anddesignated as nucleotide RT (cf. FIG. 1). The nucleotide RT wassubcloned in T vector (pBluescript KS+) and sequenced using DSQ-1000(product of Shimadzu Corp.). Thus was obtained a 2782 bp clonepolynucleotide corresponding to the full length of the murine 3T3-L1cell (ATCC No. CCL-92.1.)-derived clone 24 cDNA. This polynucleotide isdesignated as clone 24 polynucleotide.

The 100th base-involving ATG was estimated as the translation initiationcodon. As a result, the clone 24 was estimated as a gene encoding 807amino acid residues. Using the base sequence of the clone 24 asdetermined and the amino acid sequence deduced from the base sequence,homology search was carried out in the databasehttp://www.ncbi.nlm.nih.gov./BLAST/, and it was revealed that there is aChinese hamster-derived mRNA of which no function is described at alland which has been registered under the accession number AF371372 with alength of 2794 bp. Since the homology between the base sequence shownunder SEQ ID NO:1 and the base sequence of the Chinese hamster-derivedmRNA is 88.12% and the homology between the sequence from the initiationcodon to the termination codon (base No. 100 to base No. 2520 in thebase sequence) in the base sequence shown under SEQ ID NO:1 and theknown Chinese hamster-derived ORF is 91.72%, the clone 24 polynucleotideis considered to be a novel gene.

50 ng of a 5′ terminally dephosphorylated pBluescript KS+/EcoRV vectorand 150 ng of the DNA fragment were subjected to ligation reaction.Competent cells of Escherichia coli JM109 [ATCC 53323, product ofToyobo] were transformed with 1.2 μl of the ligation reaction mixture bythe heat shock method, and a subcloned clone 24 plasmid was obtained.The transformant produced in this Example 4 has been deposited with theNational Institute of Advanced Industrial Science and TechnologyInternational Patent Organism Depositary, which is an internationaldepository institution, under the accession number FERM BP-7803.Originally, the transformant was deposited with the internationaldepository institution on Nov. 29, 2000 (original date of deposition)under the accession number FERM P-18131 and, on Nov. 16, 2001, theoriginal deposit was transferred to the deposit under the BudapestTreaty.

EXAMPLE 5

Base Sequence and Amino Acid Sequence Determination

The base sequence of the clone 24 polynucleotide was determined usingABI PRISM 310 (product of Perkin-Elmer) and DSQ 1000 (product ofShimadzu). The thus-revealed base sequence of the clone 24polynucleotide is shown under SEQ ID NO:1. It was found that the clone24 polypeptide is composed of 2782 bases. Deduction of the amino acidsequence from the codon sequence of the clone 24 polypeptide indicatesthat the polynucleotide encodes the clone 24 protein composed of 807amino acid residues. The amino acid sequence of the clone 24 protein isshown under SEQ ID NO:2.

EXAMPLE 6

Comparisons with Existing Base Sequences and Amino Acid Sequences

For the base sequence of the clone 24 polypeptide and the amino acidsequence of the clone 24 protein, homology search was carried out in thedatabases Genbank, EMBL, EST and Swiss Prot.

As a result of FASTA and BLASTN, the clone 24 polypeptide shows nohomology to the polynucleotides registered with their respectivefunctions and thus was found to be a novel substance as a polynucleotidespecifically activated on the occasion of differentiation ofpreadipocytes into adipocytes.

As a result of BLASTNP, the amino acid sequence of the clone 24 proteinhas no homology to the registered proteins with respective knownfunctions, hence was found to be a novel substance as a proteinspecifically activated on the occasion of differentiation ofpreadipocytes into adipocytes.

EXAMPLE 7

Insert Recovery and Probe Preparation

The plasmid of the subcloned clone 24 obtained in Example 4 was preparedby the alkali SDS method described in Molecular Cloning, this plasmidwas cleaved with the restriction enzymes XbaI and HindIII and subjectedto 1.0% agarose gel electrophoresis, and the band corresponding to theinsert was recovered using DE81 (product of Whatman). 50 to 100 ng ofthe DNA fragment was labeled with [α⁻³²P]dCTP (product of AmershamPharmacia Biotech) using the BcaBEST™ Labeling Kit (product of TaKaRa)to give a labeled probe. The labeled probe obtained through a column ofSephadex G-50 (product of Amersham Pharmacia Biotech). A ³²P-labeledprobe was thus prepared.

EXAMPLE 8

Northern Blot Analysis

A 25-μg portion of each of the total RNAs respectively prepared frommurine 3T3-L1 cells (ATCC No. CCL-92.1.) before adipocytedifferentiation induction (0 hour) and after 0.5, 1, 3, 6, 12 and 24hours after the start of differentiation induction was subjected toelectrophoresis on a 1% modified gel (2% formaldehyde, 1×Mops, 1%agarose). The gel was treated with 50 mM NaOH for 25 minutes (alkalidenaturation) and then with 200 mM NaOAc (pH 4.0) for 40 minutes(neutralization), and the RNA was transferred to HybondN+ (product ofAmersham Pharmacia Biotech). The transfer was carried out for 12 hoursor longer. The buffer used was 20×SSC. After transfer, the filter wastreated with 50 mM NaOH for 5 minutes, washed with 2×SSC, and dried at80° C. for 2 hours, followed by UV irradiation for fixation.

Using a hybridization buffer (5×SSPE, 50% formaldehyde, 5× Denhalt's,0.1% SDS, 20 μg/ml salmon sperm DNA), prehybridization was carried outovernight at 42° C. The hybridization buffer was replaced with a freshportion, the thermally denatured probe was added, and hybridization wascarried out at 42° C. overnight. The filter was then washed with aprimary washing solution (2×SSPE, 0.1% SDS) at room temperature for 10minutes, further with the primary washing solution at 55-65° C. for 15minutes, then with a secondary washing solution (1×SSPE, 0.1% SDS) at65° C. for 15 minutes, and finally with a tertiary washing solution(0.5×SSPE, 0.1% SDS) at 65° C. for 10 minutes, and then subjected toautoradiography. The results obtained are shown in FIG. 2.

The autoradiogram shown in FIG. 2 revealed that the labeled probeprepared from the clone 24 polypeptide does not react with the mRNAprepared from the cells before the start of adipocyte differentiationinduction (0 hour) but reacts with the mRNA prepared from the cells at 3hours to 12 hours. Thus, it was found that the sequence cloned in theclone 24 polypeptide is a sequence specifically activated in adipocytedifferentiation.

EXAMPLE 9

Identification of the Site of Localization of mRNA

i) Plasmid Construction

The full length of the clone 24 polynucleotide shown under SEQ ID NO:1was amplified by RT-PCR and, after recovery and purification, theamplification product was subcloned in the GFP expression vector pEGFP(product of Clontech).

ii) Plasmid Purification

The above subcloned plasmid was subjected to two repetitions ofultracentrifugation by the Triton lysis method using cesium chloride.The supercoil plasmid DNA was thus purified.

iii) Transfection

Murine NIH/3T3 clone 5611 cells (product of JCRB 0615) which had lostthe function to differentiate into adipocytes were transfected with thesupercoil plasmid DNA obtained in the above step by the lipofectamine(product of GIBCO BRL) method. After 2 days, the transfected cells werewashed three times with PBS(−). Then, the cells were fixed by 30 minutesof treatment with 3 ml of PBS(−) containing 4% paraformaldehydecontained in a dish with a diameter of 6 cm. Then, the cells were washedtwice with PBS and, after washing, they were checked for the expressionof GFP under a fluorescent microscope (BX-50, product of OLYMPUS). FIG.3(c) is a photomicrograph showing the GFP expressed in a cell in theabove step. FIG. 3(a) is a photomicrograph of the whole cell as acontrol. FIG. 3(b) is a photomicrograph of the nucleus in the cytoplasmas stained alone by the DAPI method.

Upon comparison of the photographs (a) to (c), it is evident that theclone 24 protein-encoding mRNA occurs only in the nucleus. As to whetherthe amino acid sequence of the clone 24 protein as estimated from theclone 24 polynucleotide forms a leucine zipper structure or not,comparison with other proteins having a leucine zipper structure, namelyc-fos, C/EBPα, CREB and Jun B, indicates that they are common in thatthe leucine-to-leucine distance is 7 bases or 14 bases, hence it isestimated that also the clone 24 protein has a leucine zipper structure.That the clone 24 protein-encoding mRNA is present only in the nucleusis presumably due to the formation of a dimer via the leucine zipperstructure and its functioning as a transcription factor in the nucleus.FIG. 4 is a schematic representation of the clone 24 polypeptide. In thesequence, the sequence site considered to be a leucine zipper structureis shown in detail.

EXAMPLE 10

Production of an NIH/3T3 Cell Line Showing Excessive Clone 24 Expression

i) Plasmid Construction

The sequence from No. 87 bp to No. 2636 bp in the base sequence of theclone 24 was amplified by RT-PCR using Ampli-Taq Gold (product ofPerkin-Elmer) with an oligonucleotide having the base sequence shownbelow under SEQ ID NO:11 as a upper layer primer and an oligonucleotidehaving the sequence shown below under SEQ ID NO:12 as a lower layerprimer. After 0.8% agarose gel electrophoresis, the amplificationproduct was recovered and purified using DE81 (product of Whatman) andsubcloned in T vector (pBluescript SK+). For transformation, theEscherichia coli DH5α strain was used. After confirmation of the basesequence using DSQ-1000 (product of Shimadzu), the base sequence wascleaved with the restriction enzymes BamHI and XhoI, and the fragmentwas recovered and purified and subcloned in pDON-AI (product of TaKaRa)at the BamHI, SalI. SEQ ID NO:11 CGC AGG CCT AAG GAT GAA GGC G SEQ IDNO:12 CAG GGT CTT CTG TGG CCC TGC TCC

ii) Plasmid Preparation

Plasmid preparation was carried out using CONCERT (registered trademarkof GIBCO BRL) High Purity Plasmid Midiprep Systems (product of GIBCOBRL) and according to the manual attached thereto.

iii) Cultivation of PT67 Packaging Cells

Cultivation of PT67 packaging cells was carried out in high-glucoseDulbecco's modified Eagle medium (product of GIBCO BRL) containing 10%FBS under 5% CO₂ at 37° C.

iv) Transfection and Viral Solution Recovery

On the day before transfection, 7.0×10⁵ PT-67 cells were sowed per 10-cmplate. On the day of transfection, transfection was carried out with 14μg of pDON-AI (TaKaRa) with the full-length clone 24 inserted therein orpDON-AI containing no insert by the calcium phosphate method. At 72hours after transfection, each cell culture supernatant was recovered asa viral solution.

v) Cultivation of Target Cells NIH/3T3

Cultivation was carried out in Dulbecco's modified Eagle medium (productof Nissui Pharmaceutical Co.) under 5% CO₂ at 37° C.

vi) Viral Infection and Single Cell Line Cloning

On the day before infection, 5.0×10⁵ target NIH/3T3 cells were sowed per10-cm plate. On the day of infection, a culture fluid containing 5 ml ofthe viral solution prepared together with 8 μg/ml of polybrene (productof SIGMA) was added to the target NIH/3T3 cells. At 24 hours afterinfection, the cells were diluted stepwise at ratios 1/4, 1/10, 1/100and 1/1000 and cultured in a medium containing 0.5 mg/ml of G418(product of Nakalai Tesque) for about 10 days, and a G418-resistancecell line, namely cells with the target gene integrated into thechromosome thereof, namely an NIT/3T3 cell line showing excessive clone24 expression, was obtained.

EXAMPLE 11

Confirmation of Clone 24 Expression in Cells Produced

i) Total RNA Recovery

The total RNA was recovered from each of the NIH/3T3 cells showingexcessive clone 24 expression as obtained in Example 10 and controlNIH/3T3 cells transfected with the blank vector according to the manualattached to TRIzol (product of GIBCO BRL).

ii) Probe Preparation

The pBluescript SK+ clone 24 was cleaved with the restriction enzymePstI and, after 0.8% agarose electrophoresis, a 1.2 kbp bandcorresponding to the clone 24 cDNA was purified. The DNA fragmentcorresponding to 60 ng was labeled with [α⁻³²P]dCTP (product of AmershamPharmacia Biotech) using BcaBEST (registered trademark of TaKaRa)Labeling kit (product of TaKaRa) to give a probe.

iii) Northern Blot Analysis

The total RNA recovered (10 μg) was subjected to 1% modified gel (2%formaldehyde, 1×MOPS, 1% agarose) electrophoresis. The gel was treatedwith 50 mM NaOH for 25 minutes (alkali denaturation) and then with 200mM NaOAc (pH 4.0) for 40 minutes (neutralization treatment), and the RNAwas transferred to Hybond N+ (trademark, product of Amersham PharmaciaBiotech). The transfer was carried out for 12 hours or longer. Thebuffer used was 20×SSC. After transfer, the filter was treated with 50mM NaOH for 5 minutes, washed with 2×SSC, dried at 80° C. for 2 hours,and subjected to UV irradiation for fixation.

Using a hybridization buffer (5×SSPE, 50% formaldehyde, 5× Denhardt's,0.1% SDS, 20 μg/ml salmon sperm DNA), prehybridization was carried outovernight at 42° C. The hybridization buffer was replaced with a freshportion, the thermally denatured probe was added, and hybridization wascarried out at 42° C. overnight. The filter was then washed with aprimary washing solution (2×SSPE, 0.1% SDS) at 42° C. for 15 minutes andfurther with a fresh portion of the washing solution at 65° C. for 15minutes, and then subjected to autoradiography. The autoradiogramobtained is shown in FIG. 5.

This result revealed that the clone 24 mRNA derived from the plasmidconstructed in Example 10 i) is expressed in the NIH/3T3 cell lineshowing excessive clone 24 expression as prepared in Example 10.

EXAMPLE 12

Differentiation of NIH/3T3 Cells Showing Excessive Clone 24 Expressioninto Adipocytes

i) Differentiation Induction

The clone 24 over expressed NIH/3T3 cells obtained in Example 10 werecultured in a collagen type I dish (product of FALCON) using Dulbecco'smodified Eagle medium (product of Nissui Pharmaceutical) containing 10%calf serum under 5% CO₂ at 37° C. After the lapse of 2 days afterarrival of cells at a confluent state, when cells were in the restingphase, the medium was replaced with a differentiation inducing medium[DMEM, 10% FBS, 0.5 mM 1-methyl-3-isobutylxanthine (MIX), 10 μg/mlinsulin, 1 μM dexamethasone (DEX), BRL 49653 (product of SmithklineBeecham Pharmaceutical)], cultivation was carried out under theseconditions for 48 hours, and the medium was replaced with adifferentiation promoting medium (DMEM, 10% FBS, 5 μg/ml insulin, BRL49653). Medium exchange was carried out with the differentiationpromoting medium at 2-day intervals, and cultivation was continued today 8.

ii) Oil-Red-O Staining

5 ml of ice-cooled 4% paraformaldehyde/PBS(−) was added to the cells onthe 8th day after differentiation induction without removing the culturemedium, and the mixture was allowed to stand at room temperature for 20minutes. The medium was removed, 5 ml of ice-cooled 4%paraformaldehyde/PBS(−) was added, and the mixture was allowed to standat room temperature for 1 hour. After washing with three portions ofdistilled water, 5 ml of an Oil-Red-O staining solution (0.5% Oil-Red-O,60% 2-propanol) was added, and the mixture was allowed to stand at roomtemperature for 1 hour. The plate was washed with three portions ofdistilled water and then air-dried. The stained plate is shown in FIG.7. Upon observation under a microscope, accumulation of fatty drops wasconfirmed in the excessive clone 24 expression NIH/3T3 cells while nofatty drops were observed in the control cells. Thus, differentiationinto adipocytes was confirmed.

EXAMPLE 13

Human Homolog Cloning

i) Estimation of the Sequence of the Human Homolog Using NCBI GenomeSequencing (Human Genome Database)

For the full-length clone 24 murine polynucleotide sequence determinedin Example 7, homology searching was carried out in NCBI GenomeSequencing BLAST the Human genome(http://www.ncbi.nlm.nih.gov/genome/seq/), and a highly homologoussequence was found out on chromosome 10. When an initial methioninecodon-containing exon was regarded as the first exon, the sequence wasconstituted of 21 exons in total. Since the gt-ag rule was preservedamong all exons and introns, the full-length sequence of the humanhomolog could be anticipated.

ii) HeLa Cell Cultivation

Human homolog cloning was carried out by RT-PCR. HeLa cells were used asthe template. For HeLa cell cultivation, MEM (trademark, product ofNissui Pharmaceutical) supplemented with 10% FBS (trademark, product ofDainippon Pharmaceutical) was used.

iii) Total RNA Recovery

The total RNA was recovered from the HeLa cells cultured to a confluentstate using TRIzol (product of GIBCO BRL) and according to the manualattached thereto.

iv) Human Homolog Cloning by Reverse Transcriptase Coupled PolymeraseChain Reaction (RT-PCR)

Based on the full-length sequence anticipated in the above step i),oligonucleotides having the base sequences shown below under SEQ ID NO:3to 8 were used in combination as primers, and RT-PCR was carried outusing the RT-PCR mixture composition given below. The cDNA template wasprepared from the whole RNA of HeLa cells prepared in the above stepsii) and iii) using ReverTra Dash (product of Toyobo) and according tothe manual attached thereto. SEQ ID NO:3 (upper layer): 5′-GCC GGC ATTCAT TTA AGG CC-3′ SEQ ID NO:4 (lower layer): 5′-CTT CGC ATG AAC AGG CTCAC-3′ SEQ ID NO:5 (upper layer): 5′-CAG ATC CCA AGC TTT CGC-3′ SEQ IDNO:6 (lower layer): 5′-AGC AAA CTT GGC AAG ACC-3′ SEQ ID NO:7 (upperlayer): 5′-AAG AAG GCC CAG AGG TCA-3′ SEQ ID NO:8 (lower layer): 5′-GTCCAC TGA CTT CAT TCC-3′RT-PCR Mixture Composition:

-   2 μl 10×PCR buffer for KOD-Plus--   2 μl 12 mM dNTPs-   0.8 μl 25 mM MgSO₄-   2 μl cDNA template-   1.2 μl upper layer and lower layer primers (each 10 mM)-   11.6 μl sterile H₂O-   0.4 μl KOD-Plus-DNA polymerase (1.0 U/μl)

The primer combinations mentioned above were SEQ ID NO: 3/SEQ ID NO:4,SEQ ID NO:5/SEQ ID NO:6, and SEQ ID NO:7/SEQ ID NO:8, and the RT-PCRconditions were as follows: one cycle: 2 minutes at 94° C. 30 cycles: 15seconds at 94° C. 30 seconds at 60° C., and 2 minutes at 68° C.

v) RT-PCR Fragment Purification and Base Sequence Determination

The PCR reaction products were separated by 0.7% agarose gelelectrophoresis and then recovered and purified using DE81 (product ofWhatman). Each DNA fragment was subcloned in pBluescript KS+ andsequenced using DSQ-1000 (product of Shimadzu). The reaction productobtained with SEQ ID NO:3/SEQ ID NO:4 was not subcloned but sequencedusing Gene Rapid (product of Amersham Pharmacia Biotech).

vi) Full-Length Sequence Determination of Clone 24 Human Homolog

The clone 24 human homolog cDNA is schematically shown in FIG. 6. The394 bp band detected following RT-PCR with the combination SEQ IDNO:3/SEQ ID NO:4 was sequenced, and the band was designated as RT-1. Itwas revealed that the sequence in this region is 100% identical to thesequence anticipated in the above step i).

The 1554 bp band detected following RT-PCR with the combination SEQ IDNO:5/SEQ ID NO:6 was sequenced, and the band was designated as RT-2. Thehomology to the anticipated sequence was 1553/1554, namely 99.94%.

The 907 bp band detected following RT-PCR with the combination SEQ IDNO:7/SEQ ID NO:8 was sequenced, and the band was designated as RT-3. Itwas revealed that the sequence in this region is 100% identical to thesequence anticipated in the above step i).

The full-length nucleic acid sequence of the clone 24 human homologpolynucleotide cloned in the above manner is shown under SEQ ID NO:9.

The homology between the murine clone 24 polynucleotide shown under SEQID NO:2 and the human homolog shown under SEQ ID NO:9 was2089/2444=85.47%.

In the clone 24 human homolog polynucleotide, the 26th base-containingATG corresponding to the amino acid presumed as the first methionine inthe murine clone 24 was regarded as the translation initiation codon. Asa result, it is estimated that the clone 24 human homolog polynucleotidebe a gene encoding a protein composed of 800 amino acid residues andthat the amino acid sequence of that protein be as shown under SEQ IDNO:10.

EXAMPLE 14

Identification of Antibodies to the Clone 24

i) Introducing Maleimide Group into KLH

0.1 M sodium phosphate buffer (pH 7.0) containing 5 mg of Keyhole limpethemocyanin (trademark, product of Calbiochem, hereinafter referred to asKLH) was reacted with 1 mg of EMCS (trademark, product of DojinChemical) at 37° C. for 1 hour and, then, the unreacted EMCS was removedusing PD10 (trademark, product of Amersham Pharmacia Biotech)equilibrated with 0.1 M phosphate buffer (pH 6.0) to give maleimidatedKLH.

ii) Preparation of PEPTIDE 4-Coupled KLH

2 mg of the maleimidated KLH obtained in the above step i) was reactedwith 1 mg of Cys Lys Asp Ile Thr Pro Ser Tyr Lys Ile Arg Pro Leu Thr GluAla Glu Lys (SEQ ID NO:14), which is a product of terminally introducingCys into the sequence Lys Asp Ile Thr Pro Ser Tyr Lys Ile Arg Pro LeuThr Glu Ala Glu Lys (SEQ ID NO:13, hereinafter referred to as PEPTIDE4), which is in turn the sequence composed of 17 amino acids, i.e. fromthe 266th to 282nd amino acid, in the murine clone 24 protein shownunder SEQ ID NO:2, at 37° C. for 1 hour, and the unreacted peptide wasremoved by dialysis to give PEPTIDE 4-coupled KLH.

iii) Preparation of PEPTIDE 8-Coupled KLH

2 mg of maleimidated KLH was reacted with 1 mg of Cys Leu Arg Ile LysGlu Val Glu Val Lys Lys Asp Thr Glu Asp Ile Asn Lys Pro Lys Arg Phe (SEQID NO:16), which is a product of terminally introducing Cys into thesequence Leu Arg Ile Lys Glu Val Glu Val Lys Lys Asp Thr Glu Asp Ile AsnLys Pro Lys Arg Phe (SEQ ID NO:15, hereinafter referred to as PEPTIDE8), which is in turn the sequence composed of 21 amino acids, i.e. fromthe 426th to 446th amino acid, in the murine clone 24 protein shownunder SEQ ID NO:2, at 37° C. for 1 hour, and the unreacted peptide wasremoved by dialysis to give PEPTIDE 8-coupled KLH.

iv) Preparation of PEPTIDE 10-Coupled KLH

2 mg of maleimidated KLH was reacted with 1 mg of Cys Glu Ser Ser HisSer Lys Arg Lys Asp Lys Phe Leu Pro Gly Asp Ser (SEQ ID NO:18), which isa product of terminally introducing Cys into the sequence Glu Ser SerHis Ser Lys Arg Lys Asp Lys Phe Leu Pro Gly Asp Ser (SEQ ID NO:17,hereinafter referred to as PEPTIDE 10), which is in turn the sequencecomposed of 16 amino acids, i.e. from the 750th to 765th amino acid, inthe murine clone 24 protein shown under SEQ ID NO:2, at 37° C. for 1hour, and the unreacted peptide was removed by dialysis to give PEPTIDE10-coupled KLH.

v) Rabbit Immunization

The PEPTIDE 4-coupled KLH, PEPTIDE 8-coupled KLH and PEPTIDE 10-coupledKLH (each 50 μg) were respectively mixed with Freund's complete adjuvantto give emulsions, which were administered to rabbits dorsallysubcutaneously for immunization. After the lapse of one month from thisimmunization, booster immunization was performed using the sameemulsions and, one week later, blood was sampled from each rabbit.

vi) Preparation of Blocked, Peptide-Immobilized Solid Phase Modules

Peptide solutions were prepared by diluting, to 10 g/ml, theabove-mentioned clone 24 protein-derived peptides, namely PEPTIDE 4,PEPTIDE 8 and PEPTIDE 10, and two non-clone 24 protein-derived peptides,namely mouse-derived PEPTIDE 2 (SEQ ID NO:19) composed of 14 amino acidsand PEPTIDE 5 (SEQ ID NO:20), respectively, with PBS (product of NissuiPharmaceutical). Each peptide solution was distributed in 50-μl portionsinto wells of a 96-well titer plate module (product of Nalge NuncInternational), followed by 1 hour of incubation at 37° C. or peptideimmobilization. Then, each module was washed with PBS, 0.5% bovine serumalbumin-containing PBS was distributed in 200-μl portions into thewells, and incubation was carried out at 37° C. or 1 hour for blocking.

vii) Each rabbit anti-peptide serum prepared in the above step v) was1000-fold diluted with PBS, the dilution was distributed in 50-μlportions into the wells of the blocked, peptide-immobilized module, andthe reaction was allowed to proceed at 37° C. or 1 hour. Each module waswashed with PBS, a solution prepared by 1500-fold dilutingperoxidase-labeled goat anti-rabbit immunoglobulin (product of BiosourceInternational) with PBS was distributed in 50-μl portions into thewells, and the reaction was allowed to proceed at 37° C. for 1 hour.After washing the module, ABTS peroxidase substrate (product of KPL) wasadded, the reaction was allowed to proceed at room temperature for 10minutes, ABTS peroxidase stop solution (product of KPL) was added, andabsorbance measurements were made at 405 nm. The results thus obtainedare shown in FIG. 8.

These results revealed that the antibody obtained by immunization withPEPTIDE 4-coupled KLH reacts with the PEPTIDE 4-immobilized modulealone, the antibody obtained by immunization with PEPTIDE 8-coupled KLHreacts with the PEPTIDE 8-immobilized module alone, and the antibodyobtained by immunization with PEPTIDE 10-coupled KLH reacts with thePEPTIDE 10-immobilized module alone, but they do not react with anyother peptide-immobilized modules.

From the results of this example, it could be established thatantibodies can be obtained by immunizing rabbits with partial proteinsof the murine clone 24 protein of the invention. It is evident that whenthese antibodies are used, immunodetection systems for assaying theantigens used for immunization or proteins derived from the antigensused for immunization can be constructed.

INDUSTRIAL APPLICABILITY

The polynucleotides and proteins of the invention are respectivelypolynucleotides and proteins extractable from cells within 12 hours, inparticular around 6 hours, after the start of adipocyte differentiationinduction and are novel as polynucleotides and proteins capable of beingactivated within 12 hours from the start of adipocyte differentiationinduction.

These polynucleotides and proteins are substances very useful inelucidating the mechanisms of obesity and further properly understandingthe state of progress of obesity and utilizing the findings thusobtained in the prevention or treatment of obesity.

The polynucleotides of the invention, the proteins or peptides deduciblefrom the polynucleotides, and antibodies, antagonists or agonists to orof these peptides are useful in the treatment and diagnosis of suchlifestyle-related diseases as diabetes, hypertension andarteriosclerosis.

1. A polynucleotide encoding a protein having a function to promoteadipocyte differentiation and having a property such that it can beactivated within 12 hours after the start of adipocyte differentiationinduction.
 2. A polynucleotide as set forth in claim 1 which is apolynucleotide comprising a base sequence identical or at least 93%homologous to the base sequence shown under SEQ ID NO:1 or apolynucleotide complementary to said polynucleotide.
 3. A polynucleotideas set forth in claim 1 which is a polynucleotide comprising a basesequence identical or at least 93% homologous to that portion of thebase sequence shown under SEQ ID NO:1 which covers from the base No. 100to the base No.
 2520. 4. A polynucleotide as set forth in claim 1, whichis a DNA or RNA.
 5. An adipose differentiation promoting protein whichis a protein capable of being activated within 12 hours from the startof adipose differentiation induction.
 6. A protein as set forth in claim5 which comprises an amino acid sequence identical or at least 96%homologous to the protein specified under SEQ ID NO:2 all over the fulllength thereof.
 7. A recombinant vector containing a polynucleotidecomprising a base sequence identical or at least 93% homologous to thebase sequence shown under SEQ ID NO:1 or a polynucleotide complementaryto said polynucleotide.
 8. A transformant harboring the recombinantvector of claim
 7. 9. An antibody immunologically specific to theprotein of claim
 5. 10. A method of identifying compounds antagonizingor agonizing the protein of claim 5 which comprises: (a) bringing acandidate compound into contact with cells in which the protein of claim5 is being expressed or with cells responsive to the protein of claim 5;and (b) making observation as to the binding or the functional responsestimulation or inhibition; or comparing the cells contacted with thecandidate compound with the same kind of cells without contacting withrespect to an ability related to the activity of the protein of claim 5.11. An agonist identifiable by the method of claim
 10. 12. An antagonistidentifiable by the method of claim
 10. 13. A method of producingproteins which comprises culturing a transformant harboring arecombinant vector containing a polynucleotide comprising a basesequence identical or at least 93% homologous to the base sequence shownunder SEQ ID NO:1 or a polynucleotide complementary to saidpolynucleotide, under conditions sufficient for the formation of theprotein of claim 5, and recovering the protein from the culture mediumor the transformant.
 14. A pharmaceutical composition which comprises apolynucleotide comprising a base sequence identical or at least 80%homologous to the base sequence shown under SEQ ID NO:1 or apolynucleotide complementary to said polynucleotide.
 15. Apharmaceutical composition which comprises a polynucleotide having abase sequence comprising at least 10 consecutive bases out of apolynucleotide containing a base sequence identical or at least 80%homologous to the base sequence shown under SEQ ID NO:1 or apolynucleotide complementary to said polynucleotide.
 16. Apharmaceutical composition which comprises a polynucleotide having abase sequence comprising at least 10 consecutive bases out of apolynucleotide containing a base sequence identical or at least 80%homologous to the base sequence from the base No. 100 to the base No.2520 in the base sequence shown under SEQ ID NO:1 or a polynucleotidescomplementary to said polynucleotide.
 17. A diagnostic composition whichcomprises a polynucleotide comprising a base sequence identical or atleast 80% homologous to the base sequence shown under SEQ ID NO:1 or apolynucleotide complementary to said polynucleotide.
 18. A diagnosticcomposition which comprises a polynucleotide having a base sequencecomprising at least 10 consecutive bases out of a polynucleotidecontaining a base sequence identical or at least 80% homologous to thebase sequence shown under SEQ ID NO:1 or a polynucleotide complementaryto said polynucleotide.
 19. A diagnostic composition which comprises apolynucleotide having a base sequence comprising at least 10 consecutivebases out of a polynucleotide containing a base sequence identical or atleast 80% homologous to the base sequence from the base No. 100 to thebase No. 2520 in the base sequence shown under SEQ ID NO:1 or apolynucleotides complementary to said polynucleotide.
 20. Apharmaceutical composition which comprises the protein of claim 5 or asalt thereof.
 21. A pharmaceutical composition which comprises a peptidecomposed of at least five consecutive amino acid residues out of theprotein of claim
 5. 22. A diagnostic composition which comprises theprotein of claim 5 or a salt thereof.
 23. A diagnostic composition whichcomprises a peptide composed of at least five consecutive amino acidresidues out of the protein of claim
 5. 24. A pharmaceutical compositionwhich comprises the antibody of claim
 9. 25. A diagnostic compositionwhich comprises the antibody of claim
 9. 26. A pharmaceuticalcomposition which comprises the agonist of claim
 11. 27. A diagnosticcomposition which comprises the agonist of claim
 11. 28. Apharmaceutical composition which comprises the antagonist of claim 12.29. A diagnostic composition which comprises the antagonist of claim 12.30. A pharmaceutical composition as set forth in claim 14, which is atherapeutic agent for a lifestyle-related disease selected from amongobesity, hypertension, hyperlipidemia, diabetes, andarteriosclerosis-due heart diseases and cerebral apoplexy.
 31. Adiagnostic composition as set forth in claim 17, which is a diagnosticcomposition for a lifestyle-related disease selected from among obesity,hypertension, hyperlipidemia, diabetes, and arteriosclerosis-due heartdiseases and cerebral apoplexy.
 32. A pharmaceutical composition as setforth in claim 14, which is a therapeutic agent for obesity.
 33. Adiagnostic composition as set forth in claim 17, which is a diagnosticcomposition for obesity.
 34. A polynucleotide as set forth in claim 1which is a polynucleotide comprising a base sequence identical or atleast 89% homologous to the base sequence shown under SEQ ID NO:9 or apolynucleotide complementary to said polynucleotide.
 35. Apolynucleotide as set forth in claim 1 which is a polynucleotidecomprising a base sequence identical or at least 89% homologous to thatportion of the base sequence shown under SEQ ID NO:9 which covers fromthe base No. 26 to the base No. 2425 or a polynucleotide complementaryto said polynucleotide.
 36. A polynucleotide as set forth in claim 34which is a DNA or RNA.
 37. A protein as set forth in claim 5 whichcomprises an amino acid sequence identical or at least 93% homologous tothe amino acid sequence shown under SEQ ID NO:10 all over the fulllength of the protein.
 38. A method of identifying compoundsantagonizing or agonizing the protein of claim 37 which comprises: (a)bringing a candidate compound into contact with cells in which theprotein of claim 37 is being expressed or with cells responsive to theprotein of claim 37; and (b) making observation as to the binding or thefunctional response stimulation or inhibition; or comparing the cellscontacted with the candidate compound with the same kind of cellswithout contacting with respect to an ability related to the activity ofthe protein of claim
 37. 39. An agonist identifiable by the method ofclaim
 38. 40. An antagonist identifiable by the method of claim
 38. 41.A recombinant vector which contains the polynucleotide of claim
 34. 42.A transformant harboring the recombinant vector of claim
 41. 43. Anantibody immunologically specific to the protein of claim
 37. 44. Amethod of producing proteins which comprises culturing the transformantof claim 42 under conditions sufficient for the formation of the proteinof claim 37 and recovering the protein or a salt thereof from theculture medium or the transformant.
 45. A pharmaceutical compositionwhich comprises the polynucleotide of claim
 34. 46. A pharmaceuticalcomposition which comprises a polynucleotide having a base sequencecomprising at least 10 consecutive bases out of a polynucleotidecomprising the base sequence shown under SEQ ID NO:9 or a polynucleotidecomplementary to said polynucleotide.
 47. A pharmaceutical compositionwhich comprises a polynucleotide having a base sequence comprising atleast 10 consecutive bases out of a polynucleotide comprising the basesequence from the base No. 26 to the base No. 2425 in the base sequenceshown under SEQ ID NO:9 or a polynucleotide complementary to saidpolynucleotide.
 48. A diagnostic composition which comprises thepolynucleotide of claim
 34. 49. A diagnostic composition which comprisesa polynucleotide having a base sequence comprising at least 10consecutive bases out of a polynucleotide comprising the base sequenceshown under SEQ ID NO:9 or a polynucleotide complementary to saidpolynucleotide.
 50. A diagnostic composition which comprises apolynucleotide having a base sequence comprising at least 10 consecutivebases out of a polynucleotide comprising the base sequence from the baseNo. 26 to the base No. 2425 in the base sequence shown under SEQ ID NO:9or a polynucleotide complementary to said polynucleotide.
 51. Apharmaceutical composition which contains the protein of claim 37 or asalt thereof.
 52. A pharmaceutical composition which comprises a peptidecomposed of at least 5 consecutive amino acid residues taken out of theprotein of claim 37 or a salt thereof.
 53. A diagnostic compositionwhich contains the protein of claim 37 or a salt thereof.
 54. Adiagnostic composition which comprises a peptide composed of at least 5consecutive amino acid residues taken out of the protein of claim 37 ora salt thereof.
 55. A pharmaceutical composition which comprises theantibody of claim
 43. 56. A diagnostic composition which comprises theantibody of claim
 43. 57. A pharmaceutical composition which comprisesthe agonist of claim
 39. 58. A diagnostic composition which comprisesthe agonist of claim
 39. 59. A pharmaceutical composition whichcomprises the antagonist of claim
 40. 60. A diagnostic composition whichcomprises the antagonist of claim
 40. 61. A pharmaceutical compositionas set forth in claim 45, which is a therapeutic agent for alifestyle-related disease selected from among obesity, hypertension,hyperlipidemia, diabetes, and arteriosclerosis-due heart diseases andcerebral apoplexy.
 62. A diagnostic composition as set forth in claim48, which is a diagnostic composition for a lifestyle-related diseaseselected from among obesity, hypertension, hyperlipidemia, diabetes, andarteriosclerosis-due heart diseases and cerebral apoplexy.
 63. Apharmaceutical composition as set forth in claim 45, which is atherapeutic agent for obesity.
 64. A diagnostic composition as set forthin claim 48, which is a diagnostic composition for obesity.
 65. A methodof checking for base polymorphism which comprises applying, with thepolynucleotide shown under SEQ ID NO:9, a method selected from among thenanochip-based method, the method comprising polynucleotide sequencing,the DNA chip- or DNA array-based method, the method using a massspectrometer, and the primer extension-base method.